RESUMEN

Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior.

Asunto(s)

Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas del Grupo Polycomb/fisiología , Neoplasias de la Próstata/patología , Animales , Antígenos CD , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Transición Epitelial-Mesenquimal , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Proteínas HSP90 de Choque Térmico/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones SCID , Invasividad Neoplásica , Trasplante de Neoplasias , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Carga Tumoral

RESUMEN

Heat shock proteins (Hsps) represent a diverse group of chaperones that play a vital role in the protection of cells against numerous environmental stresses. Although our understanding of chaperone biology has deepened over the last decade, the "atypical" extracellular functions of Hsps have remained somewhat enigmatic and comparatively understudied. The heat shock protein 90 (Hsp90) chaperone is a prototypic model for an Hsp family member exhibiting a duality of intracellular and extracellular functions. Intracellular Hsp90 is best known as a master regulator of protein folding. Cancers are particularly adept at exploiting this function of Hsp90, providing the impetus for the robust clinical development of small molecule Hsp90 inhibitors. However, in addition to its maintenance of protein homeostasis, Hsp90 has also been identified as an extracellular protein. Although early reports ascribed immunoregulatory functions to extracellular Hsp90 (eHsp90), recent studies have illuminated expanded functions for eHsp90 in wound healing and cancer. While the intended physiological role of eHsp90 remains enigmatic, its evolutionarily conserved functions in wound healing are easily co-opted during malignancy, a pathology sharing many properties of wounded tissue. This review will highlight the emerging functions of eHsp90 and shed light on its seemingly dichotomous roles as a benevolent facilitator of wound healing and as a sinister effector of tumor progression.

RESUMEN

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men, and the second highest contributor of male cancer related lethality. Disease mortality is due primarily to metastatic spread, highlighting the urgent need to identify factors involved in this progression. Activation of the genetic epithelial to mesenchymal transition (EMT) program is implicated as a major contributor of PCa progression. Initiation of EMT confers invasive and metastatic behavior in preclinical models and is correlated with poor clinical prognosis. Extracellular Hsp90 (eHsp90) promotes cell motility and invasion in cancer cells and metastasis in preclinical models, however, the mechanistic basis for its widespread tumorigenic function remains unclear. We have identified a novel and pivotal role for eHsp90 in driving EMT events in PCa. In support of this notion, more metastatic PCa lines exhibited increased eHsp90 expression relative to their lineage-related nonmetastatic counterparts. We demonstrate that eHsp90 promoted cell motility in an ERK and matrix metalloproteinase-2/9-dependent manner, and shifted cellular morphology toward a mesenchymal phenotype. Conversely, inhibition of eHsp90 attenuated pro-motility signaling, blocked PCa migration, and shifted cell morphology toward an epithelial phenotype. Last, we report that surface eHsp90 was found in primary PCa tumor specimens, and elevated eHsp90 expression was associated with increased levels of matrix metalloproteinase-2/9 transcripts. We conclude that eHsp90 serves as a driver of EMT events, providing a mechanistic basis for its ability to promote cancer progression and metastasis in preclinical models. Furthermore, its newly identified expression in PCa specimens, and potential regulation of pro-metastatic genes, supports a putative clinical role for eHsp90 in PCa progression.

Asunto(s)

Transición Epitelial-Mesenquimal/genética , Proteínas HSP90 de Choque Térmico/genética , Neoplasias de la Próstata/genética , Transducción de Señal/genética , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Dipéptidos/farmacología , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Proteínas HSP90 de Choque Térmico/inmunología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Inhibidores de Proteasas/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: Perturbing Hsp90 chaperone function targets hypoxia inducible factor (HIF) function in a von Hippel-Lindau (VHL) independent manner, and represents an approach to combat the contribution of HIF to cell renal carcinoma (CCRCC) progression. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in halting the progression of advanced CCRCC. METHODS: Here we evaluated a novel next generation small molecule Hsp90 inhibitor, EC154, against HIF isoforms and HIF-driven molecular and functional endpoints. The effects of EC154 were compared to those of the prototypic Hsp90 inhibitor 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589. RESULTS: The findings indicate that EC154 is a potent inhibitor of HIF, effective at doses 10-fold lower than 17-AAG. While EC154, 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589 impaired HIF transcriptional activity, CCRCC cell motility, and angiogenesis; these effects did not correlate with their ability to diminish HIF protein expression. Further, our results illustrate the complexity of HIF targeting, in that although these agents suppressed HIF transcripts with differential dynamics, these effects were not predictive of drug efficacy in other relevant assays. CONCLUSIONS: We provide evidence for EC154 targeting of HIF in CCRCC and for LBH589 acting as a suppressor of both HIF-1 and HIF-2 activity. We also demonstrate that 17-AAG and EC154, but not LBH589, can restore endothelial barrier function, highlighting a potentially new clinical application for Hsp90 inhibitors. Finally, given the discordance between HIF activity and protein expression, we conclude that HIF expression is not a reliable surrogate for HIF activity. Taken together, our findings emphasize the need to incorporate an integrated approach in evaluating Hsp90 inhibitors within the context of HIF suppression.

Asunto(s)

Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Western Blotting , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Progresión de la Enfermedad , Impedancia Eléctrica , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Factor 1 Inducible por Hipoxia/metabolismo , Indoles , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Luciferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Panobinostat , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Genética/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

We tested whether puberty in golden hamsters is photoperiodically controlled. Hamsters were raised under 14:10 hours Light:Dark (14L) and 1:23 hours Light:Dark (1L) respectively, from birth to 28 days and tested for various parameters. Body weight, Leydig cell (LC) size and testicular testosterone secretion were greater and plasma thyroxin (T4), testicular androstenedione secretion and LC number were lower (P<0.05) in 1L than 14L hamsters. Volumes of testicular components were similar in the two groups. 3beta-hydroxy steroid dehydrogenase immunohistochemistry demonstrated LC progenitors and newly formed adult LC (ALC) in 14L hamsters, which were absent in 1L hamsters; they contained only fetal LC (FLC). Latter findings suggest the presence and absence of postnatally-differerentiated LC in 14L and 1L hamsters, respectively. Androgen results agreed with these findings, because FLC primarily secrete testosterone, and androstenedione is a major androgen secreted by the newly formed ALC. Reduced T4 in 1L hamsters is attributed to the inhibition of thyroid function by the increased duration of melatonin secretion due to non-photostimulatory conditions. The arrest in LC differentiation in 1L hamsters is attributed to low T4 levels. Although the testis size is unaltered under non-photostimulatory conditions, postnatal LC differentiation is inhibited in golden hamsters, and therefore, it is logical to suggest that their puberty is photoperiodically controlled.

Asunto(s)

Mesocricetus/fisiología , Fotoperiodo , Maduración Sexual/fisiología , Testículo/crecimiento & desarrollo , Testículo/fisiología , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Androstenodiona/fisiología , Animales , Diferenciación Celular/fisiología , Cricetinae , Inmunohistoquímica , Células Intersticiales del Testículo/citología , Masculino , Estimulación Luminosa , Radioinmunoensayo , Células Madre/citología , Testosterona/fisiología , Glándula Tiroides/fisiología , Tiroxina/sangre

RESUMEN

Previous data from our laboratory have indicated that there is a functional link between the beta-adrenergic receptor signaling pathway and the G-protein inwardly rectifying potassium channel (GIRK1) in breast cancer cell lines and that these pathways are involved in growth regulation of these cells. To determine functionality, MDA-MB-453 breast cancer cells were stimulated with ethanol, known to open GIRK channels. Decreased GIRK1 protein levels were seen after treatment with 0.12% ethanol. In addition, serum-free media completely inhibited GIRK1 protein expression. This data indicates that there are functional GIRK channels in breast cancer cells and that these channels are involved in cellular signaling. In the present research, to further define the signaling pathways involved, we performed RNA interference (siRNA) studies. Three stealth siRNA constructs were made starting at bases 1104, 1315, and 1490 of the GIRK1 sequence. These constructs were transfected into MDA-MB-453 cells, and both RNA and protein were isolated. GIRK1, ß(2)-adrenergic and 18S control levels were determined using real-time PCR 24 hours after transfection. All three constructs decreased GIRK1 mRNA levels. However, ß(2) mRNA levels were unchanged by the GIRK1 knockdown. GIRK1 protein levels were also reduced by the knockdown, and this knockdown led to decreases in beta-adrenergic, MAP kinase and Akt signaling.